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Optimization of AFLP for extremely large genomes over 70 Gb
Author(s) -
Veselá Petra,
Volařík Daniel,
Mráček Jaroslav
Publication year - 2016
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12506
Subject(s) - biology , amplified fragment length polymorphism , genome , genome size , restriction enzyme , primer (cosmetics) , intraspecific competition , genetics , gene , ecology , population , chemistry , demography , organic chemistry , sociology , genetic diversity
Here, we present an improved amplified fragment length polymorphism ( AFLP ) protocol using restriction enzymes ( Asc I and Sbf I) that recognize 8‐base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost‐effective optimization massively reduces the number of amplified fragments using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of nonidentical comigrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album . The reproducibility of this octo‐cutter protocol was calculated using several species with genome sizes ranging from 1 Gb ( Carex panicea ) to 76 Gb ( V. album ). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes ( V. album , Galanthus nivalis and Pinus pumila ).